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OP001PRECLINICAL EVALUATION OF 111In/99mTc-LABELED HUMAN GASTRIN IANALOGS IN THE DETECTION OF CCK2R+-CANCERA. Kaloudi 1, E. Lymperis 1, E. P. Krenning 2, M. de Jong 2, B. A. Nock 1, T. Maina 11. NCSR Demokritos, Athens, Greece, 2. Erasmus MC, Rotterdam, The NetherlandsE-mail: katerinakaloudi@yahoo.gr

As a part of our ongoing search toward successful localization of cholecystokinin subtype 2 receptor(CCK2R)-expressing cancer with the aid of radiopeptides (Laverman et al. 2011, Nock et al. 2005) wepresent three new radioligands: [111In]SG1 ([(111In-DOTA)Gln1,Nle15]GI, GI: pGlu-Gly-Pro-Trp-Leu-(Glu)5-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2), [

111In]SG2 [(111In-DOTA)Gln1,DGlu6-10,Nle15]GI) and[99mTc]SG6 ([(99mTc-N4)Gln

1]GI) and compare their biological profiles in CCK2R-expressing cells andmouse models. SG2 exhibited the highest binding affinity against [125I-Tyr12,Leu15]GI duringcompetition assays in A431-CCK2R(+) cell membranes (22C/1 h), followed by SG1 and SG6 (Table1). Labeling of SG1/SG2 with 111In and SG6 with 99mTc afforded the respective radioligands in highyield and high purity, as verified by RP-HPLC. All radioligands specifically internalized in A431-CCK2R(+) cells (37C/ 1 h) (Table 1). HPLC analysis of blood collected 5 min postinjection (pi) inhealthy Swiss albino mice ranked radioligands according to stability as follows: [111In]SG2 >

[

99m

Tc]SG6 > [

111

In]SG1 (Table 1). Biodistribution studies in SCID mice bearing AR4-2J xenografts at4 h pi revealed superior tumor uptake for [111In]SG1 (5.7%ID/g) followed by [99mTc]SG6 (3.3%ID/g)and the (DGlu)6-10-substituted [111In]SG2 (2.6%ID/g) (Figure 1). This trend was preserved for theCCK2R-positive mouse stomach. On the other hand, renal uptake was unfavourably high for [ 111In]SG1(Figure 1). These results demonstrate that radiopeptides based on human gastrin I sequences caneffectively target CCK2R-positive tumors in vivo. They further demonstrate that metal-chelate and Glu6-10by DGlu6-10-substitution greatly affect key biological parameters of end-radioligands, such asmetabolic stability, tumor uptake and renal clearance.

0

1

2

3

4

5

6

7

8***

/+++

*

Tumor

%ID/g

0

10

20

30

40

50

60

70

80

90

100

110

120

130

***

***/+++

Kidneys Table 1.Comparison of CCK2R affinities in A431-CCK2R(+) cell membranes, internalization in thecells and stability in mice. *non-metallated analogs, I= internalized, PP= Parent peptide.

Figure 1. Comparative uptake (%ID/g) for [99mTc]SG6 , [111In]SG1 and [111In]SG2 in AR4-2J tumor-bearing SCIDmice at 4 h pi. Statistically significant differences (*) between [111In]SG1 and (+) between [99mTc]SG6 and the otherradiopeptides: *P

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OP002STEROIDAL AROMATASE INHIBITORS IN BREAST CANCER RESEARCH. STRUCTURE ANDSUBSTRATE-GUIDED DESIGN, SYNTHESIS AND STRUCTURE-ACTIVITY RELATIONSHIPS(SAR)C. Varelaa*, F. Roleiraa, S. Costaa, R. Carvalhob, C. Amaralc,d, G. Correia-da-Silvac,d, N. Teixeirac,d, E. Tavares da SilvaaaCEF, Center for Pharmaceutical Studies & Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of Coimbra, Coimbra, PortugalbFaculty of Science and Technology & Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, PortugalcFaculty of Pharmacy, University of Porto, Porto, PortugaldInstitute for Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal

Breast cancer is the most common malignancy in women worldwide. Many breast tumors are dependent on estrogens fortheir development and growth; hence controlling estrogen production has been one of the bases for treating this disease. Thiscan be achieved by inhibiting aromatase, enzyme involved in the biosynthesis of estrogens. The recent elucidation of theactive site of aromatase, which provided structural basis for the interactions with its ligands, was an important breakthrough(Ghosh et al., 2009). This has revealed and confirmed the establishment of hydrogen bonds between the C3- and C17-ketooxygen atoms of steroids and specific residues of the active site of the enzyme. Besides, in other studies was observed that C-6 alkyl substitution can be beneficial for aromatase inhibition, since the linear side chain can protrude into an access channelimmobilizing catalytic residues.

In this work, we were interested in designing, synthesizing and evaluating the anti-aromatase activity of steroidal inhibitorsobtained by introducing C-6 methyl substituents in strong aromatase inhibitors previously reported by our group (Fig. 1)(Varela et al., 2012). Further, we were interested in investigating how the planarity in the A-ring and the establishment of aC-3 hydrogen bond are important features for aromatase inhibition.For this, we synthesize andevaluate 1, 2, 3 (1) and 4-olefins, the corresponding epoxides, and also a 3,4-cyclopropane derivative (3). Olefins were prepared by several strategies,and epoxides were synthesized by performic acid oxidation (Varela et al., 2012). Derivative3was prepared through anadaptation of the Simmon-Smith reaction. The two C-6 methyl derivatives4and5were prepared through a three- and four-step strategies.

Table 1 The anti-aromatase activity of synthetizedsteroids in placental microsomes.

Fig. 1.Structure of some synthesized androstenedione derivatives a)SEM: standard error of mean

The aromatase inhibitory activity of the modified steroids was evaluated in human placental microsomes by a radiometricassay (Varela et al., 2012). Considering the A-ring olefins and the respective epoxides, it was observed that olefins are

generally more active than epoxides, except for 2 (Varela et al., 2012). Derivative3revealed to be more active than 2 (Table1). Compounds 4and 5revealed to be very active, although slightly less potent than the corresponding derivatives withoutthe alkyl substituent (compare 4with 1, and 5with 2) (Table 1).In summary, some of the synthesized compounds are potent AIs, confirming that planarity in A,B-ring is important foraromatase inhibition. The 3,4-epoxide 2 is slightly more potent than the corresponding olefin 1allowing hypothesizing that3,4-epoxide oxygen resembles the carbonyl oxygen of androstenedione, the natural aromatase substrate (Varela et al., 2012).Nevertheless, the 3,4-cyclopropane derivative 3revealed to be even more potent than 2showing that, besides the C-3hydrogen bond, other features contribute for an efficient aromatase inhibition.

CompoundsAromastase Inhibition(%) SEMa)

IC50(M)

1 95.90 0.60 0.2252 96.40 0.10 0.1453 95.35 0.57 0.1104 93.47 1.06 0.5605 96.72 0.37 0.175Formestane 99.65 0.06 0.042

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Ghosh, D., Griswold, J., Erman, M., Pangborn, W., 2009. Structural basis for androgen specificity and oestrogen synthesis in human aromatase. Nature 457,219-224.Varela, C., Tavares da Silva, E., Amaral, C., Correia da Silva, G., Baptista, T., Alcaro, S., Costa, G., Carvalho, R.A., Teixeira, N.A.A., Roleira, F.M.F.,2012. New structure-activity relationships of A- and D-ring modified steroidal aromatase inhibitors: design, synthesis, and biochemical evaluation. J. Med.Chem. 55, 3992-4002.*Corresponding author e-mail address: carla.varela@portugalmail.com

OP004Pharmacological profile of essential oils fromAloysia citriodora Palau &Melissa

officinalis L. Relevance to neurodegenerative diseaseSawsan Abuhamdah1*, Suleiman Olimat 1 , Rushdie Abuhamdah2 and Paul.Chazot21. Faculty of Pharmacy, University of Jordan, Amman, Jordan

2. School of Biological & Biomedical Sciences, Durham University, Durham, UK

*Corresponding author: s.abuhamdah@ju.edu.jo

In traditional medical practice, numerous plants have been used to treat symptoms common in neurodegenerative diseases.A detailed pharmacological study of essential oils derived fromAloysia citriodoraPalau (Lamiaceae) leaves, cultivated inJordan and locally known as Melissa, was performed in comparison to the pharmacological properties of the essential oilfromMelissa officinalis L. (Lamiaceae), known as Melissa and cultivated in Europe.

Essential oils derived from dried and fresh leaves ofAloysia citriodora andMelissa officinaliswere analysed by GC/MS andwere investigated pharmacologically.The major components detected inAloysia citriodora oils from dried and fresh leaves included: limonene (20.1, 13.6%),geranial (6.3, 20.1%), neral (3.7, 15.1%), 1,8-cineole (9.4, 9.2%), curcumene (6.3, 3.5%), spathulenol (5.0, 3.1%) andcaryophyllene oxide (8.4, 2.2%), respectively. A number of these components were shared withMelissa officinalis, but anumber were distinct. FreshAloysia citriodora leaf essential oil inhibited [3H] nicotine binding to well washed rat forebrainmembranes (apparent mean IC50= 0.0018 mg/ml), whereasMelissaofficinalis elicited no significant effect. In contrast, theformer elicited no effects on GABAAreceptors, while the latter elicited a dose-dependent inhibition of [

35S] TBPS binding tothe GABAAreceptor (IC500.040 0.001mg/mL).Aloysia citriodora displayed concentration-dependent anti-cholinesteraseinhibitory properties, DPPH radical scavenging effect and moderate anti-oxidant activity in a ferrous chelating test (all at0.01 mg/ml and above), whileMelissa officinalis lacked these properties (up to 1 mg/ml).

we report for the first time thatAloysia citriodoraessential oils have a diverse range of

pharmacological properties, suggesting potential as asource for plant-based treatment of degenerativediseases.

Table 1: Comparative constituents of EOsderived fromAloysia citrodoraPalau. & Melissa

officinalisL.

MajorConstituents

GC-MS Analysis

Aloysia citrodoraPalau.

Melissa officinalis L.

Monoterpenoids

Limonene 13.6%1,8-cineole 9.2%Neral 15.1%Geranial 20.1%

Geranial 31.3%Neral 21.7%

Sesquiterpenoids

Caryophyllene oxide2.2%Curcumene 3.5%Spathulenol 3.1%

Caryophyllene 12.2%Caryophyllene oxide3.7%[29]

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0

20

40

60

80

100

120

140

160

50 100 250 500 1000

extract concentration (g/ml)

cellviability(%o

fcontrol)

Caco2

MCF7

3T3

Huang L, Abuhamdah S, Howes MJ, Dixon CL, Elliot MS, Ballard C, Holmes C, Burns A, Perry EK, Francis PT, Lees G,Chazot PL.,J Pharm Pharmacol.11, 1515-22 (2008). Abuhamdah S., Abuhamdah R., Howes MJ. Olimat S. & Chazot.P. Pharmacological profile of an essential oil derived fromleaves of lemon verbena (Aloysia citriodora Palau): cholinergic, anti-oxidant and iron chelation properties. J Pharm Pharmacol. (InPress).

OP005

In vitroantitumor activity of Sarcopoterium spinosumleaf extract with bioactive naturalcompoundsCeren Sunguca, Ipek Erdogana, Mehmet Emin Uslua, Oguz Bayraktarb*aIzmir Institute of Technology, Department of Biotechnology and Bioengineering Department, Izmir, Turkey

bIzmir Institute of Technology, Department of Chemical Engineering, Izmir, Turkey*Corresponding author. Address: Izmir Institute of Technology, Department of Chemical Engineering, Izmir, Turkey. Tel: +90 2327506657 Fax:

+90 2327506645.E-mail address: oguzbayraktar@iyte.edu.tr

Cancer cell lines cause generation of reactive oxygen species and free radicals at high levels (Wang and Yi,2008). Then generated free radicals lead to breakdown of the structure of DNA, lipid or protein (Gul et al., 2011).When plant extracts including antioxidant phytochemicals are exposed to the redox reactions, the harmful effectsof free radicals are effectively prevented. The aim of present research was to evaluate the antitumor potential ofthe extract derived from Sarcopoterium spinosumleaves.The leaves of S. spinosumwere collected in Izmir, Turkey. Total phenol content of ethanolic extract ofS.spinosumleaves was determined using Folin-Ciocalteu method. Total antioxidant capacity of the extract wasmeasured with ABTS+assay. The cytotoxicity of the extract on different cell lines was performed using MTTviability assay. In order to explain the results at molecular level, Real time-PCR (RT-PCR) was used. Caspase 3expression level was used as an indicator of apoptosis.S.spinosumleaf extract had significant total antioxidant capacity, along with high total phenolic content. Ourresults revealed the in vitrocytotoxic activities of S.spinosumleaf extract against different cancer cell lines andnormal cell line. The leaf extract of S.spinosumshowed promising cytotoxic activities at low concentration range,between 50 g/ml and 200 g/ml, against breast cancer cell line (MCF7) and colon cancer line (Caco2). On theother hand, it was not cytotoxic to mouse fibroblast cell line (NIH-3T3). Among cancer cell lines, the MCF7 cellline was found to be the most sensitive against the extract treatment. Cytotoxic activity was also confirmed withincreased caspase 3expression level retrieved from RT-PCR analysis. Caspase 3expression level was lowestfor 3T3 fibroblast cells. MCF7 cells were more prone to apoptosis, in the presence of extract.

References:

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Figure 1. Cell viability of S. spinosum extract-treated cell lines. Caco2, MCF7 and 3T3 represent colon cancer, breast cancerand fibroblast cell lines, respectively.

The extracts obtained from the S. spinosumleaves may represent an important source of novel potentialantitumor natural compounds due to their significant and selective cytotoxic actions towards different cancer celllines.

ReferencesWang, J., Yi, J., 2008. Cancer cell killing via ROS: To increase or decrease, that is the question. Cancer Biology& Therapy. 7, 1875-1884.Gul, M.Z., Bhakshu, L.M., Ahmad, F., Kondapi, A.K., Qureshi, I.A., Ghazi, IA. 2011. Evaluation of Abelmoschusmoschatus extracts for antioxidant, free radical scavenging, antimicrobial and antiproliferative activities using invitro assays. BMC Complement Altern Med. 11: 64.

OP008ANTI-BACTERIAL ACTIVITY OF NOVEL BIOADHESIVE OFLOXACINOCULAR INSERTSN. stndaOkur1, E. Homan Gke1, A. Yolta2, G. Ertan1, . zer11Faculty of Pharmacy, University of Ege, Bornova, Izmir, Turkey

2Faculty of Science, University of Ege, Bornova, Izmir, Turkey

The aim of the present study was to develop novel insert formulations and compare their antibacterialactivity for ocular application for treatment of bacterial keratitis.NLC formulation (F1) was prepared by means of high shear homogenization method employing oleicacid(1.456%) as oil, Compritol HD5 ATO(0.728%) as solid lipid, Tween 80(0.728%) as surfactant, andwater(97.088%). Insert formulation was prepared by means of solvent casting. F1 was mixed with0.75% chitosan oligosaccharide lactate (COL) or alginate and 5% of glycerin or PEG 400 was added asplasticizers. The mixture was poured onto the petri dishes and dried at 40C for 36h to obtain inserts.Formuations were loaded with 0.3% ofloxacin(OFX). Bioadhesion was quantified using a TextureProfile Analyzer. Minimum Inhibitory Concentration(MIC) defined as the lowest concentration ofmaterial that inhibits the growth of an organism (1), was performed with gram negative E. coliATCC8739 and gram positive methicillin susceptible S. aureusATCC 29213 the as recommended Clinical andLaboratory Standards Institute(CLSI). Microorganisms were incubated at Mueller-Hinton II Agarmedium at 37C for 17-18 hours. After the incubation, pure cultures of the microorganisms wereprepared in sterile saline solution (0.85%) and were adjusted to give an inoculum with an equivalent celldensity to 0.5 McFarland turbidity standard.1 g/mL OFX concentration were studied. 100L of sterileMueller-Hinton II broth, 100 L microorganism suspension and 100 L prepared OFX concentrationstransferred to each well and incubated for 24h at 37C. After incubation, turbidity of microplate wellswere observed. Disc diffusion method was used to evaluate antimicrobial activity of the inserts againstE. coliATCC 8739 and S. aureusATCC 29213 according to the guidelines of CLSI. Pure cultures of themicroorganisms were prepared in sterile saline solution and were adjusted to give an inoculum with anequivalent cell density to 0.5 McFarland turbidity standard. 100 l of each suspension were spreadedevenly onto Mueller-Hinton II Agar and allowed to dry. Sterile discs were then placed onto agar plates

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and 10 l of every formulation was applied to the discs. Plates were incubated at 37C for 24 to 48h andthe zone diameters of each formulation for each isolate were measured (2).Inserts which on the basis of NLC formulations, were obtained successfully.Chitosan inserts containsglycerin as plasticizer was found more bioadhesive than alginate inserts (Table 1).Table 1: Formulation components and results of bioadhesion

Formulations

Components Force (N) AUC

F2 COL (0.75%) +Glycerin

0,2930,003 1,8350,155

F3COL (0.75%) +PEG 400

0,1380,017 0,2950,022

F4Alginate (0.75%)+ Glycerin

0,1570,010 1,1350,258

F5Alginate (0.75%)+ PEG 400

0,1150,009 0,1760,018

MIC value of test inserts and OFX solution againstE. coliand S.aureuswas observed 0,390g/mL and0,781g/mL, respectively at 48h. The diameter of inhibition zone reflects magnitude of susceptibility ofthe microorganism. The chitosan acted as an anti-bacterial agent so growth inhibition ring of chitosan

insert was found 36 and 49mm against S. aureusandE. coli, respectively. The growth inhibition ring ofS. aureusandE. colitreated by alginate inserts was 35 and 40mm, respectively. The chitosan inserts hadhigher anti-bacterial activity than the alginate insertsThis study revealed that inserts containing COL showed highest anti-bacterial activity and thus could besuggested as an alternative ocular drug carrier for OFX.Reference1. Qi L et al. 2004. Carbohydr Res;339:2693700.CLSI, 2008. CLSI Document M100-S18, Clinical and Laboratory Standards Instit

OP009PACLITAXEL RELEASE FROM PH SENSITIVE LIPOSOMES: COMPARISON

BETWEEN DIALYSIS AND ULTRAFILTRATION METHODSJ. O.Eloy, J.M.MarchettiCollege of Pharmaceutical Sciences of Ribeiro Preto University of So Paulo

eloy@fcfrp.usp.br

The aims of this study were to prepare and characterize a liposomal formulation composed bydioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA) to encapsulate paclitaxel and evaluateits release, compared through the dialysis and ultrafiltration methods. Furthermore, the pH sensitivity ofthe formulation was investigated. Liposomes were prepared according to the thin-film hydrationmethod.1 Briefly, drug and lipids were solubilized in chloroform and submitted to rotaevaporation untilformation of a lipid film, which was hydrated using 8.0 pH phosphate buffer. Suspension was thenhomogenized under pressure. Liposomes were characterized by dynamic light scattering and paclitaxelencapsulation efficiency. Lipossomes were evaluated for paclitaxel release in 50 mL pH 5.0 and 7.4phosphate buffers containing 1% sodium lauryl sulfate with agitation speed at 150 rpm. In this study,samples were placed inside PVC tubes wrapped with 50 KDa MWCO (molecular weight cut-off)cellulose dialysis membranes and connected to the dissolution shafts of the apparatus 1. In theexperiments that evaluated the ultrafiltration method, samples were placed directly in the vessels,agitated with minipaddles, and the released drug was separated from encapsulated drug by ultrafiltrationusing Amicon 50 KDa MWCO ultra-2 centrifugal filter units (Millipore). Samples were collected until72 h and were analyzed by high pressure liquid chromatography, using a 25 mm C-18 column with 5 m

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particles, mobile phase with acetonitrile and water (50:50, v/v) at a flow rate of 1 mL/min andwavelength at 227 nm. Characterization studies demonstrated that the liposomal formulation exhibitednanometric size and high paclitaxel encapsulation efficiency (Table 01). Figure 01 showed that using theconventional dialysis method, paclitaxel was slowly released from the commercial solution, but wasrapidly and completed released when the ultrafiltration method was employed. The liposomalformulation released only 5% and 30% after 72 h in pHs 7.4 and 5.0, respectively, with the dialysis

method. Using the alternative ultrafiltration method, however, drug release was considerably higher,equivalent to 75% and 100% after 72 h in pH 7,4 and 5,0, respectively. Thus, the dialysis methodprovided slower release results, which could be a consequence of drug interaction with cellulose dialysismembrane, as already reported.2In conclusion, the ultrafiltration method seems to be more appropriateto evaluate paclitaxel release. Morever, the enhanced paclitaxel release from DOPE and OA liposome inacid buffer was demonstrated.

Table 01. Liposomal characterization.

Figure 01. Paclitaxel in vitrorelease

References1 YANG, T. et al. Enhanced solubility and stability of PEGylated liposomal paclitaxel: In vivo and Invitro evaluation. InternationalJournal of Pharmaceutics, v. 338, p. 317-326, 2007.2 ZAMBITO, Y., PEDRESCHI, E., DI GOLO, G. Is dialysis a reliable method for studying drug releasefrom nanoparticulate systems? A case study. International Journal of Pharmaceutics, v. 434, p. 28-34, 2012.

OP010PREPARATION , CHARACTERIZATION AND RELEASE STUDY OF MICROSPHERESLOADED WITH MYCOPHENOLIC ACID USING DIFFERENT RATIOS OF TWOMOLECULAR WEIGHT PLGA.Israa H. Al-Ani * , Alaa A. Abdulrasool** and Jabar A. Faraj

* Faculty of Pharmacy and Medical Sciences, Al-Ahliyya Amman University, Jordan,dr_issra@yahoo.com

** College of Pharmacy, Baghdad University.

Microspheres as controlled drug delivery technology has proved many advantages in controlling drugrelease for long period of time. Different types of polymers have been used in preparation ofmicrospheres with different characteristics. Biodegradable polymers offer the advantage of beingdegraded in the body to biocompatible materials, thus no need to remove the residuals after drug release.The aim of this study was to investigate the effect of molecular weight of the carrying polymer (PLGA)

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on the pattern, mechanism and time of release of mycophenolic acid from prepared microspheres usingdifferent ratios of two different molecular weight PLGA (RH202 andRH203).The microspheres were prepared by solvent evaporation method and characterized for their morphology,yield value, loading efficiency, size distribution, bulk density, degree of hydration and DSC analysis.Six batches with different ratios of both polymers were prepared as in table(1).Then the release of thedrug was studies in 37 oC phosphate buffer saline pH 7.4 using suitable dialysis cell.

Results showed that ratio of 40:60 RH202:RH203 gave the most uniform zero-order drug release over70 days in phosphate buffer saline pH 7.4 and 37oC with anomalous type of drug diffusion in addition topolymer erosion.SEM photos taken in different stages of drug release showed the gradual loss of microspheres sphericalshape which may be the cause behind the anomalous diffusion of the drug due to the non uniformhydrolysis of the polymer. However batch C which contains (40:60 RH202:RH203) could preserve zeroorder release over about 70 days. Other used ratios gave less uniform drug release that could not meetthe criteria of controlled drug delivery systems.(Figure1)In addition mycophenolic acid showed high stability in the release media since nearly 100% of its masswas recovered suggesting good stability of the prepared microspheres.It was concluded that mycophenolic acid can be loaded on two molecular weight PLGA blend

successfully with good yield value and loading efficiency and ratio of 40:60 RH202:RH203 gives zero order drug release in vitro for about 70 days in phosphate buffer saline pH 7.4Table 1 : Composition of Mycophenolic Acid Microspheres of the Prepared BatchesUsing A Mixture ofDifferent Ratios of R202H and R203H PLGA.

Batchcode

A B C D E F

% R202H% R203H

1000

8020

4060

3070

1090

0100

Figure1. Release profile of mycophenolic acid from the prepared six batches of microspheres in 0.1M

PBS pH 7.4 at 37o

C.

Key words: microspheres, PLGA, RH202 and RH203, mycophenolic acid, zero-order release, controlleddrug release.

OP012COLD ATMOSPHERIC-PRESSURE PLASMA FOR LIPOSOMAL MEMBRANEDISRUPTIONS. H. Matrali1, P. Svarnas2, F. Clment3, S. G. Antimisiaris1,41University of Patras, Department of Pharmacy, Laboratory of Pharmaceutical Technology, 26504 Rion, Greece

2University of Patras, Department of Electrical and Computer Engineering, High Voltage Laboratory, 26504 Rion, Greece

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3Universit de Pau et des Pays de lAdour, IPREM LCABIE, Plasmas et Applications, 64000 Pau, France

4FORTH/ICES, 26504 Rion, Greece (santimis@upatras.gr)

The possibility of using atmospheric-pressure cold plasma of electrical discharges instead of detergents fordisrupting liposomal membranes is investigated. The plasma system, the chemically reactive species producedand the related physical mechanisms have been considered elsewhere [Svarnas et al. (2012); Gazeli et al. (2013)].The used gas is helium (N50) flowing free in the atmospheric air.

In this work, large multilamellar vesicle liposomes (MLVs) consisting of phosphatidylcholine (PC), cholesterol(Chol) and phosphatidylglycerol (PG) are prepared by the thin film hydration technique and subjected to plasmatreatment. Liposomes encapsulate calcein (100 mM), i.e. a small hydrophilic dye whose plasma-induced releasefrom liposomes is used as a measure of liposome membrane integrity and consequently of the plasma action onthe lipid bilayers. A parametric study takes place and the principal results are depicted in Figure 1or discussedbelow.The effect of plasma treatment increases with the increase of lipid concentration. Samples with negative surfacecharge (PG) showed significant release of calcein (ca. 10% of the initial encapsulation) after only 2 min oftreatment. More calcein (up to 11%) was released from all the samples as the treatment became longer (2 to 4min). These effects that are manifested for samples with a volume of 100 L were not (or only marginally)observed when the specimen volume was doubled (200 L). On the other hand, post-treatment effect on thesamples was observed for this double volume only; the reduction immediately after plasma treatment found to be

practically 0%, but reached 7% after 48h and up to 17% after 96 hours. Both the direct and post-treatmentinfluences of the flowing gas itself were always negligible as compared to those with the plasma switched-on,under the same experimental conditions.

PC 4:1 2:1 1:1 PC:PG:Chol75

80

85

90

95

100

* ****

R

etention(%)

Liposomes' Composition

2min

3min4min

*

**** ***

* **

Figure 1:Calcein release from MLV liposomes (60 mg/mL, 100L) following plasma treatment for 2, 3 and 4 min. MLVsconsisting of PC/Chol are denoted by their PC:Chol ratio. PC:PG:Chol was 9:1:5 (mol). Significance is marked with*

(p

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Corresponding author: annika.sarnes@helsinki.fi

Ocular drug delivery is considered to be among the most challenging and fascinating research areaswithin pharmaceutical drug development, because of the complex, pharmacokinetically delicate anddemanding environment of the eye. Nanocrystal-based drug delivery systems provide efficient tools forocular formulation development, especially when considering poorly soluble drugs. The objective was toformulate ophthalmic, intraocular pressure reducing, nanocrystal suspensions from a poorly solubledrug, brinzolamide (BRA), using a rapid wet milling technique [1]. Different stabilizers (hydroxypropyl

methylcellulose (HPMC), poloxamer F127 and F68, polysorbate 80) for the nanocrystals were screened.In order to investigate both the effect of an added absorption enhancer (polysorbate 80) and the impactof the amount of free drug in the nanocrystal suspension, formulations in phosphate buffered saline(PBS) at pH 7.4 and pH 4.5 were prepared. Particle size, polydispersity (PI), solid state (DSC),morphology (SEM, TEM) as well as dissolution behavior and the uniformity of the formulations werecharacterized. The effects of nanocrystal formulations on human corneal epithelial cell (HCE-T)viability were tested. Elevated intraocular pressure (IOP) lowering effect was investigated in vivousinga rat ocular hypertensive model. Marketed BRA product was used as control throughout the study.BRA nanocrystal suspensions (450 530 nm / PI 0.1 - 0.2)were successfully prepared by wet milling inPBS pH 7.4 and pH 4.5, using HPMC as stabilizer. The final nanocrystal formulations I-III (BRA, 1w/v%; HPMC, 0.25 w/v%; benzalkoniumchloride 0.01 w/v%; with and without polysorbate 80, 0.25

w/v%) were obtained by diluting the nanocrystal suspenions with PBS pH 7.4 or pH 4.5, respectively(Table 1). Both the uniformity of the formulations and the remained crystalline state of BRA aftermilling were confirmed. The rapid dissolution of BRA (in PBS pH 7.4) from all the nanocrystalformulations was demonstrated; after one minute 100 percent of the drug was fully dissolved. The slowdissolution of unmilled bulk BRA was proven as well; only 50 percent of the drug was dissolved after30 min. Prior to thein vivo experiments the effects of the nanocrystal formulations and the marketedproduct on the cell viability were proven to be comparable. Experimentally elevated IOP reduction wasevidenced in vivowith all the formulations.The effect was significantly pronounced at pH 4.5, wherethe amount of free drug was at its highest. Notably, the experimentally elevated IOP reduction wascomparable to the marketed product. In conclusion, three ophthalmic BRA nanocrystal formulations inPBS (pH 4.5 and 7.4), which all showed advantageous dissolution and absorption behavior, were

successfully developed using a straightforward rapid wet-milling technique. The results are applicable ingeneral to the formulation development of poorly water-soluble compounds.Additionally, in contrast tothe polymeric nanoparticles, nanocrystal suspensions confer a clear regulatory advantage, since theycontain no matrix material and only consist of the drug and a comparatively small amount of stabilizer.In conclusion, the results revealed that nanocrystal suspensions are extremely potential candidates forophthalmic drug delivery and valid therapeutic approaches.

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[1] P. Liu, X. Rong, J. Laru, B. van Veen, J. Kiesvaara, J. Hirvonen, T. Laaksonen, L. Peltonen,Nanosuspensions of poorly soluble drugs: Preparation and development by wet milling, Int. J. Pharm.411 (2011) 215-222.

OP014

TARGETING HELA CELLS USING HEPATITIS B VIRUS-LIKE PARTICLE(HBVLP) DECORATED WITH NANOGLUE-CELL-INTERNALIZINGPEPTIDESK. W. Lee 1,*, B. T. Tey 3, K. L. Ho 2, B. A. Tejo 2, W. S. Tan 21. Taylors University Lakeside Campus, Subang Jaya, Selangor, Malaysia

2. Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia

3. Monash University, Bandar Sunway, Selangor, Malaysia

*khaiwooi.lee@taylors.edu.my/khaiwooi_2@yahoo.com

Recombinant viruses have been employed as a delivery vector to deliver therapeutic DNA materials anddrugs into cells. In viral vector development, retroviruses, adenoviruses, adeno-associated viruses andherpesviruses have been widely used as human gene therapy. These viruses have been geneticallyengineered to incorporate therapeutic nucleic acids of interest, and delivered into a specific host cells intheir replicative cycle. This approach is complicated and labour-intensive. Furthermore, it may have therisk of mutagenesis and oncogenesis. Nanoparticles and recombinant virus-like particle (VLP) arehighly organised structure with discrete size and shape. They can be easily manufactured in largequantity and moulded to fit specific needs via genetic or chemical modification. Numerous studies onthe employment of nanoparticles and VLPs for targeted drug and gene delivery have been reported. Therecombinant hepatitis B virus core antigen (HBcAg) produced in bacterial system self-assembles intohollow icosahedral VLP (HBVLP) which can be served as potential nanovehicle. HBVLP possessesrepetitive amino acid residues which contain functional side chains for chemical modification (Fig. 1).The modification sites can either be the natural amino acid (aa) residues or the genetically inserted aaresidues in the viral proteins. We have shown previously that the chimeric HBcAg system, displaying a

liver specific ligand at itsN-terminus was able to deliver fluorescein molecules into liver cells, in vitro(1). Therefore, it is important to investigate the common ligand display capability of the HBVLP inorder to facilitate the development of HBVLP into a cell-targeting delivery system. In this study, theHBV capsid-binding peptide (CBP; nanoglue) was employed to present HeLa cell-internalizing peptides(CIPs) on the HBVLPs. The CIP was co-synthesized at the N-terminal end of the CBP and conjugated atthe tips of the spikes of the HBVLPs by using chemical cross-linkers (CIP-HBVLPs). In order to assessthe potential of the CIP-HBVLPs as a delivery vehicle, fluorescent molecules were employed and testedon HeLa cells in vitro(2). Fluorescence microscopy showed that HBVLPs carrying the fluorescentmolecules were translocated into HeLa cells by using this method (2). This study demonstrated a proofof principle for cell-targeting delivery via CBP conjugation on HBVLPs.

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Fig. 1:The exposure of aa functional groups for HBcAg polypeptide(subtype adyw). E8and C107are notaccessible.

REFERENCES1. Lee, K. W., Tey, B. T., Ho, K. L., Tan, W. S., 2012. Delivery of chimeric hepatitis B core particlesinto liver cells.J. Appl. Microbiol.112. 119-131.2. Lee, K.W., Tey, B.T., Ho, K.L., Tejo, B.A., 2012. Nano-glue: An alternative way to display cell-internalizing peptide (CIP) at the spikes of hepatitis B virus core nanoparticles for cell-targeting

delivery.Mol. Pharm.9, 2415-2423.OP015PEO-b-PCL/DPPC chimeric nanocarriers: self-assembly aspects in aqueous and biological media and drug releasestudiesNatassa Pippa 1,2, Stergios Pispas2, Aristides Dokoumetzides1, Costas Demetzos1,*1Department of Pharmaceutical Technology, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens,Greece2Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, Athens, Greece(*)Corresponding author: Prof.Costas Demetzos; e-mail: demetzos@pharm.uoa.gr

In this work, we report on the self assembly behavior and on stability studies of mixed amphiphilic nanosystems consisting ofDPPC (dipalmitoylphosphatidylcholine) and poly(ethylene oxide)-b-poly(-caprolactone) (PEO-b-PCL) block copolymer inHPLC-grade water, phosphate buffer saline (PBS) and fetal bovine serum (FBS). These nanosystems are sterically stabilizednanovectors and can be utilized as chimeric advanced Drug Delivery nano Systems (aDDnSs) with stealth properties (Pispas2011). A gamut of light scattering techniques (static, dynamic and electrophoretic) and fluorescence spectroscopy were usedin order to extract information on the structure, morphology, size, effective charge and internal nanostructure of thenanoassemblies formed, as a function of block copolymer content, as well as temperature and concentration. Theincorporation of PEO-b-PCL leads to nanoassemblies of smaller size. All the mixed formulations were found to retain theiroriginal physicochemical characteristics for the course of two weeks. The hydrodynamic radii (Rh) of mixed nanosystemsdecreased in the process of heating up to 50C (Fig. 1). Gradual degradation of the polymeric chain in an acidic dispersionmedium, which leads to gradual structural changes of the chimeric nanovectors, was observed (Pippa et al., 2013). Themicropolarity of the hydrocarbon region of nanocarriers changed significantly in HPLC grade water and PBS with increasingblock copolymer content. The incorporation of indomethacin (IND) led to a decreased size of chimeric nanocarriers (Table1). The incorporation efficiency of mixed liposomal/block copolymer formulations for IND was increased in PBS incomparison to the HPLC-grade water, due to electrostatic interactions between drug molecule and choline headgroups. It isobserved that the in vitrorelease of the drug from the prepared chimeric nanostructures is quite fast especially for the mixed

nanovectors prepared with the lower ratio of gradient block copolymer (Pippa et al., 2013). The combination of blockcopolymers with liposomes for the development of a novel chimeric nanovector appears very promising, mostly due to thefact that the PEO-b-PCL acts as a modulator for the release rate of IND. PEO-b-PCL grafted DPPC liposomes are found tobe effective nanocontainers for the encapsulation of IND, especially at the highest molar ratio of the block copolymer.ReferencesPippa, N., Kaditi, E., Pispas, S., Demetzos, C., 2013. PEO-b-PCL/DPPC chimeric nanocarriers: self-assembly aspects inaqueous and biological media and drug incorporation. Soft Matter 9, 4073-4082.Pispas, S., 2011. Vesicular structures in mixed block copolymer/surfactant solutions. Soft Matter 7, 8697-8701.

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Fig. 1. (a) Rhand (b) dfvs. temperature for DPPC:PEO-b-PCL mixed liposomes with 0, 1, 5 and 10mol% of incorporatedblock copolymer.

CompositionDPPC:PEO-PCL :IND

Rh (nm) df % IE

9:0.1:1 32.5 2.6 10.6

9:0.5:1 34.1 2.4 11.5

9:1:1 38.4 1.9 13.5

Table 1. The physicochemical characteristics of mixed liposomes incorporating indomethacin (IND).

OP017INVESTIGATION OF ELECTROSPUN NANOFIBERS AND THEIRINFLUENCE ON CELL GROWTHJ. Pelipenko1, P. Kocbek1, J. Zupan1, J. Kristl1*1University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia*julijana.kristl@ffa.uni-lj.si

Electrospun polymeric nanofibers are gaining increasing importance in the field of biomedicine,including tissue engineering, wound healing and drug delivery applications. In tissue regenerationnanofibers mimic the fibrillar elements of natural extracellular matrix, which provides biological andphysical support for cell growth. The aim of our work was to prepare well-characterized poly(vinyl

alcohol) (PVA) nanofibers, to evaluate their properties, and to investigate the influence of their thicknessand alignmenton cell growth in order to discover the crucial properties for clinical use.Aqueous solution of PVA (Mowiol2098, Mw 125,000 g/mol) was used for preparation of electrospunnanofibers, which were subsequently thermally stabilized. Morphological properties, mechanicalcharacteristics and swelling behavior of produced nanofibers were investigated. Cells, eitherkeratinocytes or fibroblasts, were seeded on nanofibrillar support (randomly oriented or alignednanofibers) or glass coverslip used as a control. The rate of cell adhesion was determined by counting ofunattached cells, while cell migration by agarose drop assay. Proliferation was tested by MTS assay andeffect of nanofibrillar support on cell morphology was evaluated by confocal fluorescence microscopy.Additionally, the effect of PVA nanofibers on cell gene expression was determined by PCR after 3 and 6days of incubation.

Optimization of polymer solution and electrospinning conditions enable preparation of beadless PVAnanofiber, which were resistant to rapid aqueous dissolution after thermal treatment. The keratinocyteattachment rate on PVA nanofibers was determined to be lower compared to their attachment to glasscoverslip used as a control. Cell morphology was strongly influenced by nanotopography and alignmentof nanofibers. The morphology was less spread when cells were grown on randomly oriented nanofibers(Fig.1A) compared to cells growth on glass coverslip (Fig.1C). On the other hand, it was shown thataligned nanofibers can successfully direct the migration and proliferation of cells (Fig.1B). Theinfluence of the thickness of nanofibrillar supports on cell metabolic activity and morphology was alsoconfirmed. Mobility of cells grown on randomly oriented nanofibers was limited due to partial cell

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entrapment between nanofibers. Randomly oriented nanofibers increased proliferation of keratinocytes,while decreased proliferation of fibroblasts. Aligned nanofibers strongly increased proliferation ofkeratinocytes, while the proliferation of fibroblasts was comparable to the proliferation of fibroblastgrown on glass coverslip. Nanofibers significantly affected gene expression in keratinocytes andfibroblasts, at both investigated time points. To sum up, the nanofibrillar support with nanosizedinterfibrillar pores enables efficient cell proliferation and could therefore accelerate wound healing in

vivo, but it does not support 3D tissue regeneration.Our research work represents a significant step forward towards the production and application ofnanofibers in clinical practice as advanced dressings for chronic wound healing. Critical aspects ofnanofibercell interactions have been highlighted.

Fig. 1: Morphology of cells grown on different supports: (A) randomly oriented nanofibers, (B) alignednanofibers, and (C) glass coverslip as a control.

REFERENCESBhardway N., Kundu S.C. Biotechnol. Adv. 2010, 28: 325-347.Pelipenko J. et al. Eur. J. Pharm. Biopharm. 2013, 84: 401411.

OP018Decoration of NPs with a new type of curcumin-derivative and its application onA-aggregation.S. Mourtas1, E. Markoutsa,1S. G. Antimisiaris1,2

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1University of Patras, Patras, Greece;

2Institute of Chemical Engineering and High Temperatures, FORTH/ICE-HT, Patras, Greece

Among several approaches aimed at inhibiting progression of Alzheimer's disease (AD), targeting theproduction and clearance of the amyloid-beta (A) peptide is the most advanced.In order to target amyloid-beta (A) peptide our group synthesized new curcumin (curc)-derivatives and

immobilized them on nanoliposomes (NLs) [Mourtas et al. (2010); Lazar et al. (2013)]. Such approachesincrease the binding affinity of curcumin derivatives for Apeptides, due to multivalency. Herein wesynthesized a novel non-planar DPS-PEG-curc derivative (1; Table 1) and the corresponding DPS-PEG-curc surface-decorated nanoliposomes (DNLs 2; Table 1). The effect of DNLs on A1-42aggregationwas tested.For DPS-PEG2000-curc (1) synthesis, commercially available DSPE-PEG2000-maleimide was reactedwith 4-methoxytrityl-thiol / DIPEA to give the corresponding DSPE-PEG2000-S-Mmt, which waspurified by column chromatography and identified using ESI-MS and 1H-NMR. Removal of Mmt-groupin presence of 1% trifluoroacteic acid (TFA)/triethylsilane (TES) (95:5) gave the unprotected DSPE-PEG2000-SH, which was further reacted with curc in presence of DIPEA to the desired DPS-PEG 2000-curc (1). This new lipidic-curc derivative was purified and identified as above.

NLs consisting of DPPC/Chol (2:1) and 10 mol% DPS-PEG2000-curc were prepared via thin film methodto give DPS-PEG2000-curc DNLs (2: Table 1). Physicochemical characterization (particle size,polydispersity and zeta-potential) of vesicles was performed by DLS (Nano-ZS, Malvern, UK) at 25C.Table 1. Schematic representation of curcumin derivative and decorated nanoliposomes

1:DPS-PEG-curcumin 2:DPS-PEG-curcuminNLs (DNLs)

The mean diameter of DNLs was 120 nm (PDI: 0.200) and -potential was -2.03 mV The effect of DPS-PEG2000-curc DNLs on Aaggregation was evaluated by the ThioflavinT (ThT) assay. A1-42was de-seeded before the experiment using an age reversal protocol. Finally, a mixture of A1-42in Tris-HCl,ThT in Tris-HCl and DPS-PEG2000-curc DNLs [or in absence of NLs (control-1) or in presence of plainNLs (control-2)] were incubated and FI measurements were taken at specific time points. Results(Figure 1) show that DPS-PEG2000-curc DNLs (2) are able to substantially inhibit aggregation of A1-42in vitro, in the same way as the previously studied DNLs without PEG spacer [Lazar et al. (2013)],while the control NLs had no effect. Such DNLs can be further decorated with additional ligands (forbrain targeting) and explored as a novel treatment for Alzheimer's disease.

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0 20 40 60 80 1000.5

1.0

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Thioflav

in

FI(relativevalue)

Time (h)

Plain peptides (CONTROL1)LIP (CONTROL 2)DPS-curc DNLs (no PEG)DPS-PEG-curc DNLs

Figure 1. Apeptide aggregation in absence and presence of DNLs (or control NLs)

Project received funding from the European Communitys Seventh Framework Programme (FP7/ 2007-2013) under grant agreement no. 212043.

Lazar, A., Mourtas, S., Youssef, I., Parizot, C., Dauphin, A., Delatour, B., Antimisiaris, S.G., Duyckaerts, C.,2013.Curcumin-conjugated nanoliposomes with high affinity for amyloid deposites: possible applications toAlzheimer disease. Nanomedicine. 9(5), 712-721.Mourtas, S., Canovi, M., Zona, C., Aurilia, D., Niarakis, A., La Ferla, B., Salmona, M., Nicotra, F., Gobbi, M.,Antimisiaris, S.G., 2011. Curcumin-decorated nanoliposomes with very high affinity for amyloid-1-42 peptide.Biomaterials. 32, 1635-1645.

OP021

IN VIVO BIODISTRIBUTION AND INFLAMMATION-IMAGING STUDIESWITH NEW, 99mTc-LABELLED, DERIVATIVES OF THE

IMMUNOMODULATORY PEPTIDE PROTHYMOSIN C.-E. Karachaliou1, C. Triantis1, C. Liolios1, L. Palamaris2, C. Zikos1, O. Tsitsilonis3, G. Loudos2, M.Papadopoulos1, I. Pirmettis1, E. Livaniou11. National Center for Scientific Research Demokritos, Athens 15310, Greece.

2. Technological Educational Institute, Athens 12210, Greece.

3. Faculty of Biology, University of Athens, Athens 15784, Greece

e-mail:xrisak15@hotmail.com, livanlts@rrp.demokritos.gr

Prothymosin alpha (ProT) is an ubiquitously expressed polypeptide (109 amino acid long in human)exerting a dual role: an intracellular one, associated with cell proliferation and an extracellular one,associated with the enhancement of cell mediated immunity 1. According to some previous data, thedominant immunoreactive site of the molecule is located in its C-terminus, i.e. the amino acid region

100-1092

. In the present study, two specific derivatives of ProT, both containing the C-terminaldecapeptide ProT(100-109) [TKKQKTDEDD], were synthesized, purified, characterized, labelled withthe radioisotope 99mTc and used in in vivobiodistribution and imaging studies in an inflammation mousemodel, along with suitable negative control - peptides (scrambled peptides), in order to follow theradioactivity accumulated in the inflammation locus at various time intervals. More specifically,theProTpeptide-derivatives as well as the corresponding scrambled peptides were synthesized followingthe Fmoc strategy, purified with RP-HPLC and characterized with ESI-MS. Then, they were 99mTc-labelled and analyzed in terms of their radiochemical purity and stability with well-established methods.The overall yield of the peptide synthesis was > 20% and the derivatives purity was >95%. The

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radiolabelling yield was also >95%, without colloid formation. The stability tests showed no significanttranschelation of the radiometal, as well as satisfactory plasma stability.The radiolabelled peptides wereadministered in Swiss albino mice bearing experimentally induced inflammation and the mice wereeither sacrificed 2min, 30min or 2h post injection (p.i.) for biodistribution studies (organ excision andradioactivity measurement), or anesthetized for dynamic whole body imaging using a high resolutionSPECT microcamera in planar mode. Regions of interest were defined to inflamed and control organs

using the opensource ImageJ software. The biodistribution of the radiolabelled ProTderivativesdemonstrated fast clearance from the blood, heart, lungs and normal muscle. The high percentage ofradioactivity in the urine from the first 30 min p.i., combined with low activity in the liver and intestines,indicates excretion predominantly via the urinary system. The most important data is the slow clearanceof radioactivity from the inflammation locus, resulting in high contrast ratios of inflamed/control tissuefor both derivatives. The biodistribution data clearly agreed with the results of the imaging studies. Inparallel, in vitrocell-binding studies using human neutrophils are currently under way. Concluding, twonew ProTderivatives were designed, synthesized, successfully radiolabelled with 99mTc and seempromising inflammation targeting agents; moreover, the new derivatives may contribute to furtherelucidation of the multifaceted biological role of ProTin living organisms.References: 1. A. Mosoian, Future Med Chem. 2011 Jul;3(9):1199-208, 2. M. Skopeliti et al., Cancer

Immunol Immunother. 2006 Oct;55(10):1247-57OP022IMITATION OF PHASE I METABOLISM OF ANABOLIC STEROIDS BYTITANIUM DIOXIDE PHOTOCATALYSISMiina Ruokolainena, Minna Valkonena, Tiina Sikanena, Tapio Kotiahoa,b, Risto KostiainenaaDivision of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5E), FI-

00014, FinlandbLaboratory of Analytical Chemistry, Department of Chemistry, P.O. BOX 55 (A.I. Virtasen aukio 1) FI-00014, Finland

e-mail: risto.kostiainen@helsinki.fi

The most important pathways in phase I metabolism are enzyme-catalyzed oxidations. Therefore,

various oxidation methods, such as metalloporphyrins, Fenton reaction and electrochemical reactionshave been studied as alternatives for in vitro phase I metabolism studies (Lohmann and Karst 2008).Also oxidation products from titanium dioxide (TiO2) photocatalysis have been recently shown tocorrelate with metabolism products (Calza et al. 2004). The aim of this study was to further investigatethe feasibility of TiO2photocatalysis for imitation of phase I metabolism of anabolic steroids.The photocatalytic reactions of testosterone, methyltestosterone, metandienone, nandrolone andstanozolol were carried out in liquid phase using TiO2Degussa P25 particles and ultra violet (UV) light.The duration of UV exposure (225 mW/cm2) was optimized to produce maximal amount of reactionproducts. The metabolism reactions were studied in vitrousing human liver microsomes (HLM). Thesamples were analyzed with ultra high performance liquid chromatography electrospray quadrupoletime-of-flight mass spectrometry in positive ion mode.

The reaction products formed fast in TiO2photocatalysis. The optimal length of UV exposure was 2 minfor testosterone, methyltestosterone, metandienone and nandrolone, and 15 min for stanozolol, becausethere was more inhibiting acetonitrile in the reaction mixture due to poorer water solubility ofstanozolol. For all the steroids studied, the main reactions observed both in TiO2photocatalysis andHLM incubations were dehydrogenation, hydroxylation or combination of these two. Several isomers ofhydroxylation and hydroxylation+dehydrogenation products were formed in both systems. Thesimilarity of the products having the same mass and retention time in HLM and TiO2photocatalyticreactions was evaluated based on the product ion spectra. Many of the products observed in HLM

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reactions were also formed in TiO2photocatalytic reactions. However, products characteristic to eitherof the systems were also formed.In conclusion, TiO2 photocatalysis is a fast and simple method for imitation of phase I metabolismreactions. Although the main reactions were same in TiO2photocatalysis and HLM reactions, thestereochemistry of the products might be different and the feasibility of photocatalytic reactions forsimulation of drug metabolism needs to be further studied.

ReferencesCalza, P., Pazzi, M., Medana, C., Baiocchi, C., Pelizzetti, E., 2004. The photocatalytic process as a toolto identify metabolitic products formed from dopant substances: the case of buspirone J. Pharm.Biomed. Anal. 35, 919Lohmann, W., Karst, U., 2008. Biomimetic modeling of oxidative drug metabolism. Anal. Bioanal.Chem. 391, 7996

OP024Simulations investigating Bayesian dose individualization of oral BusulfanEfthtymios Neroutsos, Georgia Valsami, Aris Dokoumetzidis1

.National & Kapodistrian University of Athens, GreeceCorrespondence: Aris Dokoumetzidis, adokoum@pharm.uoa.gr

Busulfan is widely used as an alternative to total body irradiation (TBI), in the preparative regimens beforehematopoietic stem cell transplantation (HSCT) and presents a high variability, so its administration must beindividualized based on AUC. The AUC after oral Busulfan administration needs to be estimated by a Bayesianmethod, using prior information from a popPK model. Although it is best to build an in house popPK model forBayesian individualization, often prior information is obtained from literature.The aim of the present study was to investigate the usage of a popPK model from literature for Bayesianindividualization of oral Busulfan dosing in pediatric patients from a different hospital. This was performed bysimulating patients using a popPK model from literature and adjusting the dose by applying a different popPKmodel also from literature. Furthermore, a scheme including an initial i.v dose followed by regular oral busulfanadministration was studied.A total of 10000 children were simulated in NONMEM for three different blood sampling schemes, according toa PopPK model from literature (model A) [Trame et al., 2011]. Based on the posthoc estimates of clearance forthese patients using the same model (model A), the AUC was calculated and the dose was individualized targetingAUC=1125 M*min (therapeutic range of Busulfan 900-1350 M*min) while the AUC of the second day ofadministration was recorded by simulating again. Patients that fall outside the therapeutic range (TR) werecounted before and after the dose adjustment (day 1 vs day 2 of treatment). The dose of the same simulatedpatients was also adjusted by applying a different model from literature (model B) [Schiltmeyer et al., 2003] usingthe same procedure.Without dose individualization, for a blood sampling scheme at 2, 4, 6 hours, the percentage of patients whoseAUC fell within the therapeutic range was 27.8%. After individualization with model A (the same one used forsimulation) patients within therapeutic range increased to 69% while after individualization with model B (adifferent model to the one used to simulate the patients) increased to 56.5% (Figure 1). As expected better

performance was achieved with an in-house model while the overall performance even of the in-house model ismoderate. Richer sampling schemes, namely at 2, 3, 4, 5, 6 h and 1.5, 3.5, 5, 6, 8, 12 h, or initial i.v.administration followed by regular oral busulfan administration, performed similarly without offering significantimprovement (Table 1).

Table 1.% of patients falling within TR before and after dose individualization for the different sampling schemes and type ofadministration.

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Figure 1:Percent of patients within TR before and after dose individualization.The firstcolumn corresponds to the % of patients within TR without dose individualization (dose 1mg/Kg). The second and third columns correspond to the % of patients who come into the TRafter dose adjustment using model A and B, respectively.

In conclusion, in the present study we observed that Bayesian individualization of oral Busulfan dosing offerssignificant improvement while the development of an in-house model rather than the use of a literature model isdeemed necessary.References1. Trame MN, Bergstrand M, Karlsson MO, Boos J, Hempel G. Population pharmacokinetics of busulfan inchildren: increased evidence for body surface area and allometric body weight dosing of busulfan in children. ClinCancer Res. 2011 Nov 1;17(21):6867-77.2. Schiltmeyer B, Klingebiel T, Schwab M, Mrdter TE, Ritter CA, Jenke A, Ehninger G, Gruhn B, WrthweinG, Boos J, Hempel G. Population pharmacokinetics of oral busulfan in children. Cancer Chemother Pharmacol.2003 Sep;52(3):209-16.

OP025APPLICATION OF THE SIMCYPSIMULATOR IN THEPHARMACOKINETIC DRUG-HERB INTERACTION STUDY OF LOSARTANWITHRHODIOLA ROSEAMarios Spanakis1,2, Ioannis S. Vizirianakis2, Ioannis Niopas2*1Institute of Computer Science, Foundation for Research and Technology Hellas (FORTH)-Heraklion, Greece.

2Department of Pharmacognosy and Pharmacology, School of Pharmacy, Aristotle University of Thessaloniki (AUTH),

Thessaloniki, Greece

*Corresponding authors email: niopas@pharm.auth.gr, ivizir@pharm.auth.gr

In a recent work we have assessed the in vivopharmacokinetic (PK) interaction between a herbalproduct of the adaptogenRhodiola rosea (R. rosea, golden or arctic root) and losartan (1). In thiswork we have attempted to investigate through the application of a PB/PK model the potential extend ofinteraction between losartan andR. roseaextract in humans.PK experimental data were taken from published studies related to losartan and its metabolite,EXP3174, as well as fromR. rosea, and were appropriately fitted in the Simcyp v.12 simulator (SimcypLtd, Sheffield, UK) to generate a PB/PK model. The inhibitory capacity of the herbal product in PKprocesses was estimated by carrying out in vitroexperiments with recombinant CYP2C9and CYP3A4,as well as, by assessing P-glycoprotein function in Caco-2 cells. Simulations were run in population of

Samplingscheme

% patients in TRBefore doseindividualization

After doseindividualization with modelA

After doseindividualization with modelB

OralA 27.8 69.0 56.5B 28.4 70.4 56.4C 28.6 76.8 57.3IV & OralA 26.7 81.5 57.4

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healthy volunteers with simultaneous administration of single dose of losartan (50 mg) and R. roseaextract (10 mg of rosavin in which the herbal extract product was standardized). The CYP2C9polymorphisms were taken into account for the evaluation of the results. The extent of interaction wasestimated through the AUC ratio as proposed from the FDA guidance for industry.The data taken from simulations have shown a 1.42 and 1.61 mean fold increase in AUC and C maxrespectively for losartan plasma concentrations and 1.28 mean fold increase in AUC ratio of portal vein

concentrations. In addition to these results, the Cmaxand AUC ratio in plasma and portal veinconcentrations for the main losartan metabolite, EXP3174, have shown a mean reduction by 19% and28%, respectively. Differences in the AUC ratio were observed between CYP2C9polymorphisms.The results obtained from the simulations tend to propose thatR. roseaextract mainly modulateslosartans absorption and metabolism during the first-pass effect without significant influence in the PKprofile of EXP3174, which is in line with the conclusions regarding the previous in vivointeractionstudy (1). Losartan, as well as drugs with similar PK properties could be used in PB/PK models andmoreover, in PK drug-herb interaction studies where in vitro results indicate both inhibition of transportand metabolism (2). Importantly, the data obtained in this study indicate the usefulness of themethodology presented toward the pharmacological evaluation of herbal medicinal products and theapplication of Simcypplatform in assessing clinically relevant drug-herb interactions.

References1. Spanakis M, Vizirianakis IS, Batzias G, Niopas I. (2013) Pharmacokinetic Interaction betweenLosartan andRhodiola roseain Rabbits. Pharmacology, 91(1-2):112-6.2. Hellum BH, Tosse A, Hoybakk K, Thomsen M, Rohloff J, Georg Nilsen O. (2011) Potent in vitroinhibition of CYP3A4and P-glycoprotein byRhodiola rosea. Planta Med,76(4):331-8.

OP027Saliva versus Plasma Pharmacokinetics: Theory and Application of aSalivary Excretion Classification SystemNasir Idkaidek* and Tawfiq ArafatCollege of Pharmacy and Jordan Center for Pharmaceutical Research, University of Petra, Amman, Jordan

The aims of this work were to study pharmacokinetics of randomly selected drugs in plasma and salivasamples in healthy human volunteers, and to introduce a Salivary Excretion Classification System.Saliva and plasma samples were collected for 35 half-life values of sitagliptin, cinacalcet, metformin,montelukast, tolterodine, hydrochlorothiazide (HCT), lornoxicam, azithromycin, diacerhein,rosuvastatin, cloxacillin, losartan and tamsulosin after oral dosing. Saliva and plasma pharmacokineticparameters were calculated by noncompartmental analysis using the Kinetica program. Effectiveintestinal permeability (Peff) values were estimated by the NelderMead algorithm of the ParameterEstimation module using the SimCYP program. Peff values were optimized to predict the actual averageplasma profile of each drug. All other physicochemical factors were kept constant during the

minimization processes. Sitagliptin, cinacalcet, metformin, tolterodine, HCT, azithromycin, rosuvastatinand cloxacillin had salivary excretion with correlation coefficients of 0.590.99 between saliva andplasma concentrations. On the other hand, montelukast, lornoxicam, diacerhein, losartan and tamsulosinshowed no salivary excretion. Estimated Peff ranged 0.1644.16 104 cm/s, while reported fractionunbound to plasma proteins (fu) ranged 0.010.99 for the drugs under investigation. Saliva/plasmaconcentrations ratios ranged 0.1113.4, in agreement with drug protein binding and permeability. ASalivary Excretion Classification System (SECS) was suggested based on drug high (H)/low (L)permeability and high (H)/low (L) fraction unbound to plasma proteins, which classifies drugs into 4classes. Drugs that fall into class I (H/H), II (L/H) or III (H/L) are subjected to salivary excretion, while

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those falling into class IV (L/L) are not. Additional data from literature was also analyzed, and all resultswere in agreement with the suggested SECS. Moreover, a polynomial relationship with correlationcoefficient of 0.99 is obtained between S* and C*, where S* and C* are saliva and concentrationdimensionless numbers respectively. The proposed Salivary Excretion Classification System (SECS)can be used as a guide for drug salivary excretion. Future work is planned to test these initial findings,and demonstrate SECS robustness across a range of carefully selected (based on physicochemical

properties) drugs that fall into classes I, II or III.

(1) Amidon, G. L.; Lennernas, H.; Shah, V. P.; Crison, J. R. A theoretical basis for a biopharmaceutic drug classification: the

correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 1995, 12 (3), 413420.(2) Guidance for Industry: Bioavailability and Bioequivalence Studies for Orally Administered Drug Products

GeneralConsiderations. March 2003. Division of Drug Information, Center for Drug Evaluation and Research, Food and Drug Administration

5600 Fishers Lane Rockville, MD 20857, USA.

OP028DEVELOPMENT AND VALIDATION OF AN ELISA METHOD FORCETUXIMAB QUANTIFICATIONR. Petrilli, C. S. Bitencourt, N. Najar, R. F. V. LopezUniversity of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil

raquelp@fcfrp.usp.br

Cetuximab is a chimeric monoclonal antibody direct against epidermal growth factor (EGFR), a knownmarker for squamous cell carcinoma1(SCC). The use of cetuximab is currently restricted to systemictherapy leading to various side effects. In this context, we purpose the topical application of cetuximabfor skin SCC. Therefore, indirect ELISA quantifications were performed using carrier free EGFR asantigen (BD biosciences), Blocker Blotto in TBS: Blocker Casein in TBS (20:80 v/v) (ThermoScientific, USA) as plate blocker and a mix between HRP conjugated and unconjugated antibody (1:5,v/v) as the secondary antibody. TMB solution (Invitrogen, USA) was added to detect complex betweendrug and secondary antibody and the reaction was stopped using hydrochloric acid 1M. The yellowcolor was read using a plate reader2(Multiskan FC, Thermo Scientific, USA) at 450 nm. The methodoptimization was proceeded by testing different concentrations of antigen EGFR (0.5, 1.0, 1.5 and 1.75g/mL) and of detection antibody (from 0.5to 4.0 g/mL). Cetuximab dilutions were tested in differentmedia (water, PBS 100 mM pH 7.4, citrate buffer 30 mM pH 6.0 and assay buffer). The concentrationsof coating and detection antibody chosen for method validation were those able to provide the greatestsignal:noise ratio, without blank background. The method was validated using ICH Q2-R1 guideline.EGFR carrier free concentration and antibody detection concentrations were optimized for 1.75 g/mLand 4.0 g/mL, respectively. Cetuximab dilutions in water showed a better profile than for the bufferstested and were chosen for method validation and for future liposomal preparations. Lower limit ofquantification was determined as 0.125 g/mL. Linearity was obtained in the range of 0.125 g/mL to0.75 g/mL with R>0.99. Intra-day and inter-day precision and accuracy were tested in all concentrationlevels (n=6) and revealed CV values under 15% for precision and recovery between 87.3% and 107.2%

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for accuracy. Based on the results, a method for cetuximab quantification by ELISA was developedusing EGFR at 1.75 g/mL in PBS for plate coating and detection antibody at 4.0 g/mL. The methodwas validated in the range of 0.125 to 0.75 g/mL with adequate linearity, accuracy and precision.

0.0 0.2 0.4 0.6 0.80.0

0.2

0.4

0.6

Y= 0,6446x + 0,070R= 0,997

Concentration (g/mL)

Absorbance(n

m)

Figure 1. Standart curve for cetuximabe solutions from 0.125 to 0.75 g/mL.

Table 1. Precision and accuracy of the methodConcentration(g/mL)

Intra-dayPrecision(%)

Intra-dayAccuracy(%)

Inter-dayPrecision(%)

Inter-dayAccuracy(%)

0.125 14.6 96.5 14.4 99.00.25 13.8 101.8 13.6 101.40.375 7.6 103.3 7.9 107.20.5 16.2 104.6 13.9 93.60.75 10.3 87.3 8.3 94.0

References1 Chng et al. (2008). Epidermal growth factor receptor: a novel biomarker for aggressive head and neck

cutaneous squamous cell carcinoma. Hum. Path. 39, p. 344-349.2 Hantash, J. et al (2009). The development, optimization and validation of an ELISA bioanalyticalmethod for determination of Cetuximab in human serum. Anal. Methods 1, p. 144-148.

PP005SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL ACTIVITY OFHYDROXAMIC ACIDS DERIVED FROM OLIVE OIL TRIACYLGLYCERIDESM. Barbari1, . Marini2, D. Viki-Topi2, M. Zovko Koni1, K. Mikovi3, M. Baus Lonar 2,3, M. Jadrijevi-MladarTaka1*1Faculty of Pharmacy and Biochemistry, Zagreb, Croatia;

2Institute Ruer Bokovi, NMR Center, Zagreb, Croatia;

3

School of Medicine, Osijek, Croatia

* Corresponding author: mjadrijevic@pharma.hr

Fatty hydroxamic acids (FHAs), obtained from olive oil triacylglycerides by hydroxylaminolysis (Hoidyet al., 2010) (Fig. 1) were evaluated for their biological activity, i.e.against radical scavenging activity(RSA), metal chelating activity (ChA), antioxidant activity (AOA) in -carotene-linoleate assay, FHAsreducing power (RP), as well as for their cell toxicity on normal (fibroblast) cell line (BJ) and tumourcell line (HeLa).Elemental analysis, IR and 1H and 13C 1D NMR spectroscopy data indicate that the obtained productsare predominantlyN-oleoyl hydroxamic acids (OHA) with some percentage ofN-linoleyl hydroxamic

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acid (LHA). This was additionally confirmed by HMQC and HMBC 2D NMR spectra and MALDI-TOF /TOF mass spectrometry.

Fig.1. The reaction equation of fatty hydroxamic acids (FHAs) from olive oil.

The results of in vitroassays of FHAs showed notable antioxidant, radical-scavenging and chelatingproperties (Table 1).

Table 1. The results of biological activitytesting of FHAs

The cell toxicity testing revealed that FHAsaffected more normal cells (fibroblasts) byreducing their growth while the effect on fastgrowing tumor cell line was mild.N-oleoyl hydroxamic acid (OHA) and itscorrespondig carboxylic acid (oleic acid,OA) were analysed by using Molinspirationsoftware engine v2011.06, with the aim ofprediction of bioactivity scores for the most

important drug targets. The following drug-likeness scores were computed: for OHA, the enzymeinhibitor (0.55) and protease inhibitor (0.58) and for OA the enzyme inhibitor (0.27) and the nuclearreceptor ligand (0.23).ADMET PredictorTM 6.5 calculated properties predicted the OHA is CYP 2E1 substrate, and OA asnon-substrate. The predicted ADMET risk and TOX MUT risk of OHA are 2.0 and its TOX risk 0.0,while predicted parameters for OA are 4.0, 0.0 and 1.0, respectively. The results of biological activitytesting and computed data for FHAs spotlight OHA as a promising lead-compound for further research.Reference: Hoidy W.H., Ahmad M.B., Jaffar Al-Mulla E.A., Zin Wan Yunus W.M., Bt Ibrahim N., 2010. Synthesis andCharacterization of Fatty Hydroxamic Acids from Triacylglycerides. J. Oleo. Sci. 59, 15-19.

PP007ESSENTIAL BIOMETALS (Fe, Zn, Mg, Mn, K) IN THE TEA MIXTURES FORTHE TREATMENT OF NUTRITIONAL ANEMIA IN THE BALKANPENINSULA (SERBIA)R. Nikoli1, N. Krsti1*, V. Dimitrijevi1, I. Arsi2, J. Jovanovi3, M. Nikoli11Faculty of Sciences and Mathematics, University of Nis, Serbia; 2Faculty of Medicine, University of Nis, Serbia; 3Highmedical school, uprija, Serbia; Corresponding author: nenad.krstic84@yahoo.com

Biometals are carriers of vital functions of the body and they are part of different enzymes and otherbioactive complexes that are required for the performance of vital processes. Change of biometalconcentration can lead to a variety disorders. The required dailiy amount of biometals enter the bodymainly by food. One of disorders caused by the deficiency of biometals is nutritional anemia. (Hussainet al., 2006; Tokaliolu, 2012) In this study was determined the content of some essential biometals in

SampleRSA EC50(g/ml)

ChA EC50(g/ml)

AOA EC50(g/ml)

RPEC0.5(g/ml)

FHAs235.66 54.01a* 1226.53 58.33b*

55.71 1.29a*

395.71

47.37c*

Standard 24.46 2.54a

12.64 2.81b

1177.96 175.73d45.61 0.11a

97.02810.87c

Values (g/ml) are means SD (n = 3); Standards: aButylatedhydroxyanisol, bEDTA,cAscorbic acid, dQuercetin; *Statistical differences with thecorresponding standards (P< 0.05)

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tea mixtures that are traditionally used for the treatmant of nutritional anemia in the Balkan Peninsula(Serbia).Plant species that are used for the preparation of tea mixtures are Stinging nettle (Urtica dioica L.),Sideritis scardica (Sideritis scardica Griseb.J Lamiaceae) and Rosa canina (Rosa canina L). The plantmaterial was dried, burned (t = 110-150 C) and then annealed at temperatures up to 500 C. After that itwas mineralizaed (conc. HNO3/conc. HCl) and, finally, the dry residue dissolved in deionized water.

Concentration of essential biometals (Fe, Zn, Mg, Mn, K) in the te mixtures were determined byinductively coupled plasma-optical emission spectrometry (ICP-OES).The results presented in Figure 1, showed that tea mixtures which were selected contain relatively highamount of Fe (0.9-1.9 mg/g). Stinging nettle also contains large amount of K and Mg (25.1 mg/g K i 3.3mg/g Mg).

Figure 1:Concetration of biometals in the tested plant species (a) and % of the daily requirement (d.r.)of biometals in a cup of tea (n=5, n-number of samples) (b)

Based on the results of the study the selected tea mixtures can be used as supplements in the treatment ofnutritional anemia due to the high concentration of Fe. By consuming a cup of tea we also intake otheressential biometals. Using Stinging nettle as anjuct to vegetable in meals can increse the intake ofbiometals up to half of daily needs.Acknowledgements: This work was supported by the Ministry of Education. Science andTechnological Development of the Republic of Serbia under the Project No. III45017.

References: Hussain, I., Khan, F., Khan, I., Khan, L., Walli-Ulah, 2006. Determination of heavy metalsin medicinal plants. Jour. Chem. Soc. Pak. 28(4), 347-351.Tokaliolu, S., 2012. Determination of trace elements in commonly consumed medicinal herbs by ICP-MS and multivariate analysis. Food Chem. 134, 2504-2508.

PP008DESIGN, SYNTHESIS AND EVALUATION OF NEW STEROIDAL 17-CARBOXY DERIVATIVES AS 5-REDUCTASE INHIBITORSC. Varelaa, E. Tavares da Silvaa, C. Amaralb,c, G. Correia-da-Silvab,c, R. Carvalhod, S. Costaa, S. Cunhae, J. Fernandese, N.Teixeirab,c, F. Roleiraa*aCEF, Center for Pharmaceutical Studies & Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of

Coimbra, Coimbra, PortugalbFaculty of Pharmacy, University of Porto, Porto, PortugalcInstitute for Molecular and Cell Biology (IBMC), University of Porto, Porto, PortugaldFaculty of Science and Technology & Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra,PortugaleREQUIMTE, Faculty of Pharmacy, University of Porto, Porto, Portugal

The androgens testosterone (T) and dihydrotestosterone (DHT), besides playing an important role inprostate development and growth, are also responsible for the development and progression of benignprostate hyperplasia (BPH) and prostate cancer. Therefore, the actions of these hormones can be

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antagonized by preventing the irreversible conversion of T into DHT, inhibiting 5-reductase. Steroidal5-reductase inhibitors (RIs), finasteride and dutasteride, are used in clinic for BPH treatment and havebeen also proposed for chemoprevention of prostate cancer. Nevertheless, they still promote bone andmuscle loss, impotency and occurrence of high-grade prostate tumors. Hence, it is important to searchfor other potent and specific molecules with lower side effects.In this work, new steroidal RIs were designed and synthesized by structural changes at the C-17

carboxy group of 4-androsten-3-one-17-carboxylic acid 1, an analog of 5-reductase substrate (T), withcarboxamide and carboxyester functions, through the reaction of the acid with the respective amine, indichloromethane, using triethylamine, dimethylformamide and the coupling reagent BOP (Fig. 1). Thecarboxyester 5was synthesized without the amine reagent. The inhibitory activity was evaluated inhuman prostate microsomes using a new methodology that was developed by our group, based ondispersive liquidliquid microextraction followed by gas chromatographymass spectrometry (Amaralet al., 2013a).The antiproliferative effects were studied in a human androgen-responsive prostate cancer cell line(LNCaP cells) (Amaral et al., 2013b). It was observed that the C-17carboxylic acid 1is a weakinhibitor (Table 1). Among the amides synthesized and evaluated, the N-tert-butylcarboxamide (2)(molecule combining the A-ring of the substrate T and the C-17carboxamide of finasteride) showed

the best activity. Regarding derivatives withN-propylcarboxamide (3) andN-hexylcarboxamide (4)groups, these revealed to be also strong inhibitors. However, they were less active than derivative 2showing that a hinderedN-alkyl group in the C-17carboxamide derivative favors the inhibitoryactivity.Concerning the carboxyester derivative (5), it revealed only moderate activity. It was alsoobserved that carboxamide derivatives allowed a decrease in the viability of stimulated LNCaP cells in a5-reductase dependent way, being even more effective than finasteride. (Amaral et al., 2013b)In summary, taking into account the studied compounds, it is possible to conclude that the C-17lipophilic carboxamide group along with the 3-keto-4moiety in the A-ring seem to be favorable keyfeatures for achieving 5-reductase inhibitory activity, being the C-17N-tert-butylcarboxamidederivative the best RI. Furthermore, the most potent steroids synthesized revealed to induce a decreasein the viability of stimulated LNCaP cells in a 5-R dependent-manner, being the synthesized steroidseven more effective than finasteride. This study can help the future design of new steroidal RIs, withfewer side effects.

Amaral, C., Cunha, S.C., Fernandes, J.O., Tavares-da-Silva, E., Roleira, F.M.F., Teixeira, N., Correia-da-Silva, G., 2013a. Development of a new gas chromatography-mass spectrometry (GCMS)methodology for the 5-reductase activity. Talanta 107, 154-161.Amaral, C., Varela, C., Correia-da-Silva, G., Tavares-da-Silva, E., Carvalho, R.A., Costa, S.C.P.,Cunha, S.C., Fernandes, J.O., Teixeira, N., Roleira, F. M.F., 2013b. New steroidal 17-carboxyderivatives present anti-5-reductase activity and anti-proliferative effects in a human androgen-responsive prostate cancer cell line. Biochimie, in press.

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Corresponding author e-mail address: froleira@ff.uc.pt

C

O

OOH

3

N

H

C

CH3

CH3

CH3

N

H

(CH2)2CH3

2 4 N

H

(CH2)5CH3

O N

NN

C

O

OR

5

R =

R =

R =

R =

34

5

1

RNH2Et3N

17

DMF, BOP,CH2Cl2

Fig. 1.General reaction to prepare the studied compounds as RIs

Table 1 The anti-5-reductase activity of synthetized steroids in human prostate microsomes.

Compounds Reductase Inhibition(%) SEM

a)

IC50(M)

1 29.55 3.09 -2 73.08 3.05 0.373 69.33 1.03 0.46

4 63.56 1.08 0.615 49.20 2.51 -Finasteride 84.62 1.21 0.096

a)SEM: standard error of mean

PP009SYNTHESIS, IMMUNOMODULATORY ACTIVITIES AND MOLECULARCALCULATIONS IN GROUP OF ISOXAZOLE DERIVATIVESM. Mczyski1, J. Artym2, M. Kociba2, M. Zimecki2, E. Drozd-Sczygie1, A. Sochacka-wika1, A. Koll3, S. Ryng11 . Wroclaw Medical University, Borowska 211a, 50-556 Wrocaw, Poland2. Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocaw, Poland3

. University of Wrocaw,

14F Joliot-Curie str., 50-383 Wrocaw, Poland

The aim of this report was to select a most biologically active compound and proceed molecularcalculations among a series of substituted benzylamides of 5-amino-3-methyl-4-isoxazolecrboxylic acid.The described derivatives were synthesized in reaction of substituted benzylamines with 5-amino-3-methyl-4-isoxazolecarboxylic acid azide. The immunological methods encompassed: determination ofphytohemagglutinin A (PHA)-induced human peripheral blood mononuclear cell (PBMC) proliferation,cytokine production by human whole blood cell cultures, humoral immune response of mousesplenocytes in vitroto sheep erythrocytes (SRBC), cellular immune response in mice in vivotoovalbumin (OVA), and inflammatory response to carrageenan in mice.The compounds exhibited differential, but generally immunosuppressive properties in the applied tests.MO5 compound was selected in in vivoexperiments as the most active in inhibition of: PHA-inducedcell proliferation, humoral immune response and tumor necrosis factor (TNF ) production.Interestingly, MO5 stimulated the inductive phase of the cellular immune response but strongly inhibitedthe effector phase of this response. The compound inhibited also the carrageenean reactionthatconfirmed its strong anti-inflammatory character. In summary, MO5 combines anti-proliferative andanti-inflammatory activities, and its effect on the humoral and cellular immune responses is differential.The molecular calculations revealed that the isoxazole ring plays an important role in the observedimmunological activities. The differences in the observed immunosuppressive properties of the studiedderivatives of isoxazole are a good reason for the theoretical investigations. The performed ab initio

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calculations provided useful information on the electron charge distribution in described molecules. Theisoxazole ring is common part of all studied compound and can be considered as the reference molecularsubunit. The charge distribution of the isoxazole ring should be related with the electronic structure ofwhole molecule.Due to itsinteresting and beneficial properties, MO5 compound should be further investigated in moreadvanced models. The molecular calculations suggest that distribution of atomic charges at synthesized

derivatives may condition its distinct immunoregulatory nature.The study was supported by grant of Polish National Science Centre nr N N405 682840.

PP010EXEMESTANE POTENTIAL METABOLITES DESIGN, SYNTHESIS AND STUDIES INBREAST CANCER CELLSC. Varelaa, F. Roleiraa, A. Lopesb,c, C. Amaralb,c, G. Correia-da-Silvab,c, N. Teixeirab,c, E. Tavares da Silvaa*aCEF, Center for Pharmaceutical Studies & Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of Coimbra, Coimbra,

PortugalbFaculty of Pharmacy, University of Porto, Porto, Portugal

cInstitute for Molecular and Cell Biology (IBMC), University of Porto, Porto, Portugal

Exemestane (Aromasin) is the only steroidal aromatase inhibitor (AI) that is orally active and highly potent leading to anirreversible inhibition of the enzyme aromatase. It is used in the treatment of hormone-responsive breast cancer inpostmenopausal women. Exemestane is extensively metabolized in the body and, as observed for many drugs, the resultingmetabolites can be active compounds. Therefore, we were interested in synthesizing potential metabolites, structurally relatedto exemestane, by substitution of double bonds by epoxide functions, since epoxidation reactions are proposed in themetabolic pathways for metabolizing exemestane.Epoxide derivatives were synthesized from exemestane (Figure 1) by two kinds of oxidative reactions. For derivative2, itwas used performic acid and for 3, hydrogen peroxide in alkaline medium, as oxidants. Concerning the 17-hydroxyderivative 1, it was obtained as a by-product from the reaction with the reductive mixture of trifluoroacetic acid, acetic acid,acetonitrile and sodium borohydride, in an attempt to reduce the C3-carbonyl group.The prepared compounds were evaluated in placental microsomes by a radiometric assay (Varela et al., 2012). Their effectsin cell proliferation and cell viability in breast cancer MCF-7aro cells were also studied, according to reported methods(Amaral et al., 2013).

Regarding the epoxides (2and 3), substitution of the C-6 exocyclic and C-1/C-2 double bonds by the epoxide function led tovery potent derivatives, being the compound resulting from the exocyclic substitution (2) slightly more potent than theresulting from substitution at C-1/C-2 (3).The C-17-hydroxy derivative (1), although being less active than exemestane, isalso a strong AI. All the synthesized compounds revealed to induce a decline in cell viability and cell-proliferation in a time-and dose-dependent manner.

Figure 1. Structure of exemestane derivativesIn summary, results revealed that, according to that we have already observed for other steroids tested as AIs, the chemicalsubstitution of a double bond by an epoxide function allows obtaining very strong AIs. Further, the substitution of the C-17carbonyl group by the hydroxyl group leads to a slightly decrease in potency. In addition, the potential metabolites inhibitcell proliferation even more efficiently than exemestane, showing that other mechanisms of cell death, in addition toaromatase inhibition, may be involved.

Amaral, C., Varela, C., Azevedo, M., Tavares da Silva, E., Roleira, F.M.F., Chen, S., Correia-da-Silva, G., Teixeira, N., 2013. Effects ofSteroidal Aromatase Inhibitors on Sensitive and Resistant Breast Cancer Cells: Aromatase Inhibition and Autophagy. J. Steroid Biochem.135, 51-59.Varela, C., Tavares da Silva, E., Amaral, C., Correia da Silva, G., Baptista, T., Alcaro, S., Costa, G., Carvalho, R.A., Teixeira, N.A.A.,Roleira, F.M.F., 2012. New Structure-Activity Relationships of A- and D-Ring Modified Steroidal Aromatase Inhibitors: Design,Synthesis, and Biochemical Evaluation. J. Med. Chem. 55, 3992-4002.

*Corresponding author e-mail address: etavares@ff.uc.pt

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PP012ASSESMENT OF THE NITROGEN AND COMPOST DIFFERENT LEVELS EFFECTS ONQUALITATIVE AND QUANTITATIVE PERFORMANCE OF Calendula officinalis L.Amin rezazadeh sarabi, *, Parisa Farahpour1 , Morteza sam deliriAgriculture department, Islamic Azad University of Chalous, Chalous, Iran

*E-mail:Aminrezazade.agc@gmail.com

Recently, the harmful side effects of chemical drugs on human health cause that researchers focus onMedical herbs more than last decades. Nitrogen has a basic effect on qualitative and quantitativeperformance of plants. Calendula officinalis L is a medicinal plant from Astraceae family. Severalstudies revealed that fertilizers specially nitrogen increases the yield amount of medical plants; however,it may reduce its healing effects. So, assessing the effect of fertilizers on qualitative and quantitativeperformance of medical plants are very important. In order to investigate the effects of nitrogen andcompost different levels on qualitative and quantitative performance of Calendula officinalis L. herb, anexperiment was carried out in the research field of Chalous Azad University in 2011-2012. Theexperiment was done in factorial form as a randomized complete block design, in three replicates.

Treatments consisted of nitrogen and compost. Considered nitrogen levels consisted of N0= 0, N1=50,N2=100 kg/ha and compost levels were including C0=0, C1=6, C2=12 ton/ha. Investigated characteristicsconsisted of flower dry weight, number of flowers in plant, flower diameter, flavanoid content. Datawere analyzed by MSTATC software and Mean of characters was compared by one-way ANOVA testand 2-sided Duncan test for post hoc multiple comparison (in 8 harvest levels). The results were shownin table-1. Nitrogen and compost treatments had statistically significant influence (p0.01) on studiedcharacteristics. Flower dry weight, flower diameter and number of flower in plant characteristics hasbeen studied in eight harvest; as, the performance of these characteristics had increasing procedure fromthe first harvest up to the forth harvest; and, in the forth harvest, it has reached to its` maximum leveland from fifth harvest, it had decreasing procedure. As, up to the forth harvest, the maximum flower dryweight, flower diameter and number of flower in plant obtained by C1N2 (C1=6 ton/ha compost and

N2=100 kg/ha nitrogen treatment) and from fifth up to the eighth harvest, it was obtained by C2N2(C2=12 ton/ha compost and N2=100 kg/ha nitrogen) treatment. Also, the maximum flavonoid contentobtained by C2N1(C2=12 ton/ha compost N1=50 kg/ha nitrogen) treatment. In conclusion, applicationof compost as a biological fertilizer plays an effective role in enhancement of quantitative performanceand increment of the flavonoid content of the plant (1, 2).Table-1: Comparison the mean of different characteristics of Calendula officinalis L, treated by different levels of Compostand Nitrogen fertilizers in 8 harvests.Characteristics Treatment

Type1

st

Harvest2

nd

Harvests3

rd

Harvest4

Th

Harvest5

th

Harvest6

th

Harvest7

th

Harvest8

th

HarvestNumber ofFlowers inPlant

C0N0 3.00 5.33 7.66 14.67 14.00 12.33 7.66 2.06C1N1 13.00 15.67

25.33 30.67 28.67 22.00 11.33 6.67

C2N2 11.00 14.67 22.67 26.67 29.33 23.67 15.67 10.66

Flower DryWeight